High-level expression and characterization of a thermostable xylanase mutant from Trichoderma reesei in Pichia pastoris

• Published in: Protein expression and purification 2015-04, Vol.108, p.90-96
• Main Authors: Li, Yang-yuan, Zhong, Kai-xin, Hu, Ai-hong, Liu, Dan-ni, Chen, Li-zhi, Xu, Shu-de
• Format: Article
• Language: English
• Subjects: Endo-1,4-beta Xylanases - biosynthesis; Endo-1,4-beta Xylanases - chemistry; Endo-1,4-beta Xylanases - genetics; Endo-1,4-beta Xylanases - isolation & purification; Enzyme Stability; Fermentation; Fungal Proteins - biosynthesis; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - isolation & purification; Gene Expression; Hypocrea jecorina; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Point Mutation; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Trichoderma - enzymology; Trichoderma - genetics; Trichoderma reesei; Xylanase
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2014.11.014

•A thermostable T. reesei xylanase mutant was expressed and characterized in P. pastoris.•Optimization expression of Mxyn2 was processed by controlling pH and temperature.•High cell density fermentation of Mxyn2 was performed with optimal parameter. A gene encoding xylanase 2 mutant from Trichoderma reesei (T2C/T28C, named mxyn2) was cloned into the Pichia pastoris X33 strain using the vector pPICZαA. Recombinant Mxyn2p was functionally expressed in P. pastoris X33 and secreted into the supernatant. Real time qPCR demonstrated that an increase in gene copy number correlated with higher levels of expression. Supernatant from methanol induced cells was concentrated by ultrafiltration with a 10kDa cut off membrane, and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. Recombinant Mxyn2p protein had the highest activity at 75°C, while recombinant protein encoded by the “wild type” xylanase gene xyn2, also expressed in Pichia, was 20°C lower. The Mxyn2p enzyme retained more than 70% of its activity after incubation at 80°C for 10min. The effects of the optimal pH and temperature for higher expression levels in P. pastoris were also determined, 6.0 and 22°C, respectively. The maximum xylanase activity of Mxyn2p was 13,000nkat/mg (9.88g/l) in fed-batch cultivation after 168h induction with methanol in a 50l bioreactor.